Tuning the Cell-Adhesive Properties of Two-Component Hybrid Hydrogels to Modulate Cancer Cell Behavior, Metastasis, and Death Pathways

This work presents a polysaccharide and protein-based two-component hybrid hydrogel integrating the cell-adhesive gelatin-tyramine (G-Tyr) and nonadhesive hyaluronic acid-tyramine (HA-Tyr) through enzyme-mediated oxidative coupling reaction. The resulting HA-Tyr/G-Tyr hydrogel reflects the precise chemical and mechanical features of the cancer extracellular matrix and is able to tune cancer cell adhesion upon switching the component ratio. The cells form quasi-spheroids on HA-Tyr rich hydrogels, while they tend to form an invasive monolayer culture on G-Tyr rich hydrogels. The metastatic genotype of colorectal adenocarcinoma cells (HT-29) increases on G-Tyr rich hydrogels which is driven by the material’s adhesive property, and additionally confirmed by the suppressed gene expressions of apoptosis and autophagy. On the other hand, HA-Tyr rich hydrogels lead the cells to necrotic death via oxidative stress in quasi-spheroids. This work demonstrates the ideality of HA-Tyr/G-Tyr to modulate cancer cell adhesion, which also has potential in preventing primary metastasis after onco-surgery, biomaterials-based cancer research, and drug testing.

Condensation reaction was initiated by introducing EDC (24 mM, Sigma-Aldrich, USA), NHS (10 mM, Sigma-Aldrich, Germany) and tyramine hydrochloride (58 mM, Sigma, Germany) and pH was adjusted to 6.0. Reaction was allowed to proceed for 12 hours, the final solution was diluted with Dulbecco's phosphate-buffered saline (DPBS), and the solution was dialyzed through a dialysis membrane with 12-14 kDa cut-off to remove byproducts and unreacted reagents. After lyophilization, the product was kept at +4 o C until further use.

FT-IR spectroscopy
G-Tyr was chemically characterized by FT-IR. The spectra of pure gelatin powder and lyophilized G-Tyr were obtained with a Shimadzu IRAffinity-1 model spectrometer between 400-4000 cm -1 spectral wavelength, and the region of interest (400-2000 cm -1 ) was presented in Figure S1. The gelatin backbone was identified according to corresponding bands such as; the peak in the wavelength at 3300 cm -1 shows the -OH stretching vibrations of hydrogenbonded hydroxyl groups on the polymer; peak at 3000-3100 cm -1 shows the N-H stretching of amide-A bonds; the peak at 2920-2950 cm -1 indicates the asymmetric aliphatic C-H stretching vibrations; the peak at 2850 cm -1 belonging to symmetric aliphatic C-H stretching vibrations; sharp peaks at 1600-1630 cm -1 and 1500-1535 cm -1 indicate the amide-I and amide-II β-sheet structure, respectively. In addition, peaks at 1386 cm -1 and 1469 cm -1 represent the symmetric and asymmetric bending vibrations of methyl groups on the gelation backbone. On the other hand, same bands were observed in the spectra of G-Tyr. Additionally, distinct signal locating between 3300-3500 cm -1 wavelength in G-Tyr confirms the success in conjugation of aromatic phenol unit of tyramine with carboxyl group of gelatin. 1 However, this peak was not observed in unmodified gelatin.

Figure S3
Macroscopic image illustrating the cross-linked and un-crosslinked G-Tyr G-Tyr (5% and 10% wt.) was dissolved in DPBS untill the homogenity was ensured. HRP (2 U/mL) was added into G-Tyr solution and H 2 O 2 (2mM) was added to trigger an orthogonal enzymatic cross-linking resulting in the formation of dityramine bridge.

Scanning electron microscopy (SEM)
The microstructure of G-Tyr hydrogels (5% and 10% wt.) was investigated under SEM, which exhibited a microporous architecture ( Figure S4). As expected, the pore size of G-Tyr (~33 µm in 10% wt.) was found to be lower as compared to G-Tyr (~50 µm in 5% wt.) resulting from a dense matrix structure. This opportunity to tune matrix density enables to control matrix stiffness, thereby, cell behaviors such as adhesion, migration, and invasion will be able to control utilizing the developed hydrogels. 3,4 Figure S4. SEM micrograph of G-Tyr hydrogels

Degredation test
For the degradation test, hydrogels (5% and 10% wt.) were dehydrated overnight by freezedryer, weighted (w o ), incubated in DPBS for 1, 2, 5, and 7 day, and the lyophilized hydrogels were weighted to record the final mass (w f ). The degradation rate was calculated using the equation below: Degradation test of G-Tyr hydrogels (5% and 10% wt.) revealed that G-Tyr (5% wt.) maintained its susceptibility to degradation up to 7 days (Figure S5), while G-Tyr (10% wt.) preserved about 70% of its weight after 7-days incubation. After 7-days of degradation period, the hydrogels kept their integrity with 0.4% weight loss everyday. The gels were observed to preserve their integrity for 77 days (approximately 2.5 months) without being completely degraded. It should also be noted that incubation time of cell culture study in this work was 2days, and G-Tyr in both 5% and 10% wt. preserve its weight (>75%) after 2-days of incubation. Figure S5. Degredation test of 5% and 10% G-Tyr hydrogels

Equilibrium water intake (swelling) analysis
For the swelling test, the freze-dried hydrogels were weighted (w 1 ), incubated in deionized water for 3 hours to allow complete water intake, and the hydrogels were weighted again in swollen state (w 2 ). The water intake capacity was evaluated using the following equation: Water intake (%) = (w 2 -w 1 /w 2 ) x 100 Equilibrium water intake is an important feature of hydrogels to get knowledge about their water affinity, permeability and diffusion capacity which are important characteristics for the application of hydrogels. 5 Swelling percentages of hydrogels were yielded as 89.53% (5% G-Tyr) and 90.23% (10% G-Tyr), respectively ( Figure S6). There was no significant difference between the swelling characteristics of 5% and 10% G-Tyr hydrogels. Figure S6. Equilibrium water intake (swelling) graphs of 5% and 10% G-Tyr hydrogel (*p>0.05)

Optical transparancy measurements
To determine the transparency of G-Tyr hydrogels, hydrogels (5% and 10% wt.) were placed into deionized water containing cuvette and UV absorbance was recorded between 200-700 nm wavelength. Results demonstrated that both hydrogels absorbed the light in UV-region with the wavelenght ranges between 200-350 nm; whereas, G-Tyr hydrogels transmitted light in visible region beyond 350 nm ( Figure S7). This shows that light absorbtion in the visible region was low that almost no light was absorbed, while transmittance was high. Obtained results proved the transparancy of G-Tyr (5% and 10% wt.) hydrogels.  Eppendorf. The total volume of the tube (hexane+hexane-impregnated sample) was considered V2. The volume of the tube when hexane-impregnated sample is removed was noted as V3.
The porosity (ε) was calculated utilizing the equation 1 and given in Table 1.  The calculated porosity levels were presented in Figure S9.

Section 6: Gene expression study
The list of primers used in gene expression studies can be found in Table S2 and 3.

Table S2
Primer sequences related to mechanotransduction, EMT, and metastasis genes The obtained metabolome data was statistically processed by t-testing and ANOVA test ( Figure   S10 and 11). The pathways affected by up/down-regulated metabolites were presented in Table S4 and S5.

Table S4
Affected pathways in HT-29 cells that were cultured atop HA-Tyr and G-Tyr hydrogels